Pacific Biosciences of California, Inc.

Applied Biosystems, LLC

Life Technologies Corporation

Congcong Ma

Stephen Wilson Turner

Cheryl Heiner

Kevin Travers

Geoff Otto

Mark F Oldham

Linda G Lee

Sam L Woo

Richard T Reel

David Cox

Jon M Sorenson

Benjamin G Schroeder

Willy K Wiyatno

Paul Wyatt

Willy Wiyatno

David M Cox

Various embodiments of the teachings relate to a system or method for sample preparation or analysis in biochemical or molecular biology procedures. The sample preparation can involve small volume processed in discrete portions or segments or slugs, herein referred to as discrete volumes. A molecular biology procedure can be nucleic acid analysis. Nucleic acid analysis can be an integrated DNA amplification/DNA sequencing procedure.

Apparatus, system, and method using immiscible-fluid-discrete-volumes

Methods and systems for single molecule sequencing using concatemers of copies of sense and antisense strands. Concatemers are provided, for example, by carrying out rolling circle amplification on a circular molecule having sense and antisense regions to produce repeated copies of the sense and antisense regions connected by linking regions. The circular molecules can be produced by ligating hairpin adapters to each end of a double-stranded nucleic acid having a sense and antisense strand. The ligations can be carried out, for example using blunt end ligation. In some cases, a single molecule consensus sequence for a single template molecule is obtained. A single read from each template molecule can be obtained by comparing the sequence information of the sense and antisense regions.

Sequencing using concatemers of copies of sense and antisense strands

Computer systems, computer readable media, and computer methods for obtaining, calling, and assembling nucleic acid sequences are presented. In some aspects the invention includes the sequencing of template constructs that comprise double stranded portions in partially contiguous constructs, to provide for single molecule consensus sequence determination through one or both of sequencing sense and antisense strands in the same molecule.

Methods and systems for obtaining a single molecule consensus sequence

Methods obtaining a single molecule consensus sequence for a single template molecule, and for obtaining a plurality of single molecule consensus sequences for a plurality of single template molecules is provided. Template molecules having two complementary regions connected with a linker are sequenced. A single read from each template molecule can be obtained, the read containing sequence information for each of the complementary regions. Single molecule consensus sequences can be determined from these reads by comparing the sequence information of the two complementary regions.

Methods for obtaining a single molecule consensus sequence

Compositions and methods for nucleic acid sequencing include template constructs that comprise double stranded portions in a partially or completely contiguous constructs, to provide for redundant sequence determination through one or both of sequencing sense and antisense strands, and iteratively sequencing the entire construct multiple times. Additional sequence components are also optionally included within such template constructs. Methods are also provided for the use and preparation of these constructs as well as sequencing compositions for their application.

Compositions and methods for nucleic acid sequencing

The present invention provides compositions and methods for reducing steric hindrance in the product of nucleic acid polymerase reaction. Methods and compositions of the invention encompass application of exonucleases, endonucleases, and uracil-DNA glycosylases to a nucleic acid polymerase reaction such that newly formed nucleic acid strands are modified (e.g., cleaved) while the polymerase reaction continues to proceed.

Reaction mixtures for prevention and alleviation of steric hindrance during single molecule synthesis

Methods, substrates, and systems for diagnostic sequencing are provided. Small circles of nucleic acids from about 10 bases to about 200 bases can be sequenced, for example using template dependent sequencing by synthesis. The use of small circles of nucleic acids allows for repeated sequencing of the same portions of the nucleic acid, providing for higher accuracy sequence determinations.

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Foster City, CA, United States of America

Congcong Ma